Bacillus anthracis, the spore-forming causative agent of anthrax, generally infects herbivores (Hanna, 1998). Human infection, while rare, can result in a generally benign, self-limiting cutaneous disease or a systemic disease that rapidly leads to death in a high percentage of cases. The cutaneous disease can arise when spore particles from soil or animal products are introduced into cuts or skin abrasions. In contrast, the systemic disease can arise when B. anthracis spore particles are inhaled (LD50≈10,000 spore particles). The high mortality rate and the ability to readily prepare and deliver B. anthracis spore particles as an aerosol have made B. anthracis a dreaded agent of biowarfare and bioterrorism.
The causative agent of the systemic disease is anthrax toxin (AT), which itself comprises a pair of binary, AB-type toxins—lethal toxin and edema toxin (Leppla, 1995). Each is assembled at the surface of mammalian cells from proteins released by B. anthracis. Lethal toxin, assembled from Protective Antigen (PA, 83 kDa) and Lethal Factor (LF, 90 kDa), is primarily responsible for lethality (Friedlander, 1986; Hanna et al., 1992; Hanna et al., 1993). Edema toxin, assembled from PA and Edema Factor (EF, 89 kDa), causes edema at the site of injection (Leppla, 1982). EF has calmodulin-dependent adenylate cyclase activity. LF is a Zn++-dependent protease that cleaves certain proteins involved in signal transduction and cell cycle progression (MAPKK1 and MAPKK2) (Duesbery et al., 1998).
In these AB-type toxins, PA is the receptor-binding B moiety that delivers either EF or LF, as alternative enzymic A moieties, to the cytosol of mammalian cells (Leppla, 1995). Initially, PA binds specifically, reversibly, and with high affinity (Kd≈1 nM) to a cell-surface AT receptor (ATR). After binding to the receptor, PA is cleaved by a member of the furin family of proprotein convertases, which removes a 20 kDa fragment, PA20, from the N-terminus (Klimpel et al., 1992; Novak et al., 1992). The complementary fragment, PA63, remains receptor-bound and spontaneously self-associates to form heptameric ring-shaped oligomers (Milne et al., 1994) that avidly and competitively bind EF and/or LF (Leppla, 1995) to form EF/LF-PA63 complexes. These complexes are trafficked to an acidic compartment by receptor-mediated endocytosis. In the acidic compartment, the PA63 heptamers (the “prepore”) are inserted into the membrane, forming transmembrane pores (Gordon et al., 1988). Concomitantly EF and LF are translocated across the membrane to the cytosol. Consistent with the pH dependence of translocation, toxin action is inhibited by lysosomotropic agents and bafilomycin A1 (Mendard et al., 1996).
EF translocation causes a large increase in intracellular cAMP concentration (Gordon et al., 1988; Gordon et al., 1989). Increased cAMP levels cause edema, and in neutrophils, inhibit phagocytosis and oxidative burst (O'Brien et al., 1985). By protecting the bacteria from phagocytosis, edema toxin apparently aids in establishing bacterial infection and proliferation in the mammalian host.
Treatment of primary macrophages and certain macrophage cell lines with lethal toxin causes cell lysis (Friedlander, 1986). Macrophage-depleted mice are resistant to treatment with lethal toxin, suggesting that macrophages are the primary targets of lethal toxin (Hanna et al., 1993). Low doses of lethal toxin induce the production of interleukin-1 and tumor necrosis factor (Hanna et al., 1993). Thus, it has been suggested that hyperproduction of cytokines causes death of the host by inducing systemic shock. How these or other proteins lead to cytokine production and macrophage lysis remains unclear.
In the past few years considerable progress has been made toward a detailed understanding of the structure and function of PA. Crystallographic structures of PA and the PA63 heptamers have been determined (Petosa et al., 1997). The prepore undergoes a major conformational change under acidic conditions to form a 14-strand transmembrane β-barrel pore (Benson et al., 1998; Miller et al., 1999). The pore structure and the detailed mechanism by which LF and EF are translocated across membranes are under intensive investigation.
The ATR structure is heretofore unknown, but is present in all cell lines that have been tested. Studies on CHO-K1 cells had indicated that PA binds to a proteinaceous receptor that is present in about 104 copies/cell (Escuyer and Collier, 1991). The paucity of knowledge about the ATR represents a major gap in the understanding of how AT acts. Identification and cloning of the ATR will provide more treatment strategies for anthrax.
A cDNA clone (Genbank Accession Number NM 032208) known as tumor endothelial marker 8 (TEM8) is known (St. Croix, 2000). TEM8 is upregulated in colorectal cancer endothelium, but heretofore the function of TEM8 was not known.